Recombinant dengue virus DNA fragment

ABSTRACT

A recombinant protein encompassing the complete envelope glycoprotein and a portion of the carboxy-terminus of the membrane/premembrane protein of dengue 2 virus was expressed in baculovirus as a protein particle. The recombinant protein particle was purified and found to provide protection against lethal challenge with dengue 2 virus in mice.

INTRODUCTION

This invention relates to the production and purification of arecombinant protein for use as a diagnostic tool and as a vaccineagainst Dengue virus.

Dengue (DEN) viruses are human pathogens with a significant threat toworld health. These viruses are estimated to cause several hundredthousand cases of dengue fever, dengue hemorrhagic fever (DHF) anddengue shock syndrome (DSS) annually (Shope, R. E. In: The Togaviruses.Schlesinger, R. W. (Ed.) Academic Press, New York. 1980, pp. 47-82;Monath, T. P. In: The Togaviridae and Flaviviridae, Schlesinger, S. andSchlesinger, M. J. (Eds.) New York and London, 1986, pp. 375-440;Halstead, S. B. Bull. W.H.O. 1980, 58, 1-21; Halstead, S. B. Am. J.Epidemiol. 1984, 114, 632-648) The complete content of all documentscited herein are hereby incorporated by reference. Dengue viruses aremembers of the family Flaviridae and are transmitted by Aedes mosquitoes(Halstead, S. B. Science 1988, 239, 476-481). There are four serologicaltypes, DEN-1, DEN-2, DEN-3 and DEN-4, distinguishable bycomplement-fixation assays (Sabin, A. B. and Young, I. A. Proc. Soci.Exp. Biol. Med. 1949, 69, 291-296), virus plaque-reductionneutralization tests (Russell, P. K. and Nisalak, A. J. Immunol. 1967,99, 291-296) and immunoassays using monoclonal antibodies (MAbs)(Gentry, M. K. et al. Am. J. Trop. Med. Hyg. 1982, 31, 548-555; Henchal,E. A. et al. Am. J. Trop. Med. Hyg. 1982, 31, 830-836).

Dengue viruses are composed of a single-stranded RNA molecule ofpositive polarity (messenger sense) which is contained within anucleocapsid composed of capsid (C) protein. The capsid is surrounded bya lipid envelope about 50 nm in diameter in which are embedded theenvelope (E) glycoprotein and the matrix (M) protein. Both thestructural and nonstructural (NS) proteins are encoded by a single, longopen reading frame of about 10.5 kilobases arranged as follows:C-PreM/M-E-NS1-NS2A-NS2B-NS3-NS4A-NS5 (Rice, C. M. et al. Science 1985,229, 726-733; Wengler, G. et al. Virology 1985, 147, 264-274; Castle, E.et al. Virology 1986, 149, 10-26; Zhao, B. et al. Virology 1986, 155,77-88; Mason, P. W. et al. Virology 1987, 161, 262-267; Mackow, E. etal. Virology 1987, 159, 217-228; Sumiyoshi, H. et al. Virology 1987,161, 497-510; Irie, K. et al. Gene 1989, 74, 197-211).

Attempts to prevent DEN virus infection have focused on the productionof a vaccine which would protect against all four serotypes. However,despite more than 50 years of effort, safe and effective dengue virusvaccines have not been developed. Candidate vaccines currently beingtested fall into two categories: live attenuated dengue virus vaccinesand subunit vaccines, each with its own drawbacks.

Live attenuated virus vaccines have been demonstrated to be eitherunder-attenuated (cause disease) or over-attenuated (fail to immunize).Even an optimally-attenuated live virus vaccine can revert to a virulent(disease-causing) form through mutation. Live dengue viruses are alsosensitive to heat, making it difficult and costly to maintain thevaccine in some tropical and subtropical countries where the vaccine maybe needed most.

Recombinant subunit vaccines have the advantage of eliminating the riskof infectivity and greater chemical stability. However, the subunitvaccines of flavivirus structural and NS proteins produced in expressionvectors including baculovirus, vaccinia virus and E. coli reported sofar elicit only low titers of neutralizing antibody and are difficult toproduce in large quantities and pure form (Putnak, J. R. et al. Virology1988, 163, 93-103; Putnak, J. R. et al. Am. J. Trop. Med. Hyg. 1991, 45,159-167; Zhang, Y. M. et al. J. Virol. 1988, 62, 3027-3031; Lai, C. J.et al. In: Vaccines, Modern Approaches to New Vaccines IncludingPrevention of AIDS (Eds. Lerner, R. A. et al.), Cold Spring HarborLaboratory Press, New York, 89, 1989, pp. 351-356; Bray, M. et al. J.Virol. 1989, 63, 2853-2856; Bray, M. and Lai, C. J. Virology 1991, 185,505-508; Men, R. et al. J. Virol. 1991, 65, 1400-1407; Mason, P. W. etal. Virology 1987, 158, 361-372; Mason, P. W. et al. J. Gen. Virol.1989, 70, 2037-2049; Mason, P. W. et al. J. Gen. Virol 1990, 71,2107-2114; Murray, J. M. et al. J. Gen. Virol, 1993, 74, 175-182;Preugschat, F. et al. J. Virol 1990, 64, 4364-4374).

Both the envelope (E) and the nonstructural protein 1 (NS1) arecandidates for recombinant, subunit vaccines against DEN virus. The Eglycoprotein is the major surface protein of the virion. It functions invirion attachment to host cells and it can be detected by its ability tohemagglutinate goose erythrocytes. As an antigen, it containsvirus-neutralizing epitopes (Stevens, T. M. et al. Virology 1965, 27,103-112; Smith, T. J. et al. J. Virol 1970, 5, 524-532; Rice, C. M. andStrauss, J. H. J. Mol. Biol. 1982, 154, 325-348; Brinton, M. A. In:Togaviridae and Flaviridae. Schlesinger, S. and M. J. Schlesinger(Eds.), M. J. Plenum, New York, 1986, pp. 327-365; Heinz, F. X. Adv.Virus Res. 1986, 31, 103-168; Westaway, E. G. Adv. Virus Res. 1987, 33,45-90; Hahn, Y. S. et al. Arch. Virol. 1990, 115, 251-265). Neutralizingantibodies, believed to correlate with protection, andhemagglutination-inhibiting (HI) antibodies develop following naturalinfection. Mice immunized with purified DEN-2 E antigen developneutralizing antibodies and are protected against lethal virus challenge(Feighny, R. J. et al. Am. J. Trop. Med. Hyg. 1992, 47, 405-412).

Recombinant DEN proteins have been produced using the baculovirus systemfor the purpose of developing a vaccine. Results have been variable andsometimes disappointing. Several strategies have been used to producethe DEN E protein in the baculovirus system. One strategy used atruncated gene to produce the E protein without the hydrophobictransmembrane segment of the carboxy terminus. The purpose of thisapproach was to promote secretion and solubility of the protein.Proteins produced in this manner were minimally immunogenic in mice(Putnak, R. et al. Am. J. Trop. Med. Hyg., 1993, 45: 159-167; Zhang, Y.M. et al., J. Virol., 1988, 62: 3027-3031). Another strategy used apolygene that encoded the capsid, premembrane and two nonstructuralproteins, C-prM-E-NS1-NS2 (Delenda et al. J. Gen. Virol, 1994, 75:1569-1578). This construct produced the full length E protein bycleavage of the polyprotein. Neutralizing antibody to the full length Eprotein was not elicited by that product although protection wasinduced. The complex nature of the construct precludes an analysis ofthe reason for protection in the absence of neutralizing antibody butthe presence of NS1 in the construct was speculated to have induced theprotective response. Another strategy employed a construct thatcontained a polygene encoding C, preM and a truncated E protein (Deubelet al. Virology, 1991, 180: 442-447). Although the truncated E reactedwith some E-specific monoclonal antibodies (mAbs), reactivity was weakerthan that obtained with native virus.

Therefore, in view of the problems with the presently available vaccinesdiscussed above, there is a need for a DEN vaccine that elicits veryhigh titers of neutralizing antibody, provides protection against thedisease, has no possibility of infectivity to the immunized host, can beproduced easily in pure form, and is chemically stable.

SUMMARY

The present invention is directed to a subunit vaccine that satisfiesthis need. The recombinant DEN virus subunit vaccine of the presentinvention comprises the full dengue virus envelope protein, expressed inbaculovirus and capable of self-assembling into a particle. Dengueenvelope protein has been expressed in the baculovirus system by others.The previously produced products were poorly immunogenic when tested inanimals. None of the previously made products are known to formparticles. The protein is expressed and purified as a particle composedof multiple dengue envelope protein molecules. Particles are moreimmunogenic than soluble proteins, possibly because they can crosslinkcell surface immunoglobulins on B cells. The envelope protein particleof the present invention is produced in baculovirus in large quantitiesand in pure form, elicits high titers of neutralizing antibody and isprotective against the disease in the immunized animal.

The present invention describes the production of the DEN envelopeprotein particle by cloning the complementary DNA (cDNA) sequencesencoding the envelope protein fragment into an expression vector suchthat the recombinant dengue protein can be expressed. The recombinantprotein is produced in baculovirus, isolated and purified as a particlewhich is antigenic, reactive with dengue virus-specific and monoclonalantibodies and capable of eliciting the production of neutralizingantibodies when inoculated into mice. The administration of thisrecombinant subunit vaccine is demonstrated to protect mice, an acceptedanimal model, against morbidity and mortality following challenge withlive dengue virus.

Therefore, it is an object of the present invention to provide a DEN 2cDNA fragment encoding the full envelope glycoprotein, said genecontaining 1485 nucleotides plus 93 adjacent upstream sequences andextending from 844 to 2422 of the viral genome and is useful as adiagnostic agent and a naked DNA vaccine.

It is another object of the invention to provide a recombinant vectordesigned to produce the recombinant DEN envelope protein for use as avaccine and as a diagnostic agent.

It is still another object of the invention to provide a purified DENenvelope protein particle useful as a vaccine against DEN disease andfor detecting the presence of said disease in a suspected patient.

It is another object of the present invention to provide a method forthe purification of recombinant DEN envelope protein particle for use asa vaccine or as a diagnostic tool.

It is yet another object of the invention to provide a DEN virus vaccineeffective for the production of antigenic and immunogenic responseresulting in the protection of an animal against dengue virus disease.

BRIEF DESCRIPTION OF THE DRAWINGS

These and other features, aspects, and advantages of the presentinvention will become better understood with reference to the followingdescription and appended claims, and accompanying drawings where:

FIGS. 1. (A-C) Illustration of the pBlueBacIII shuttle vector and genesequences used for expression of the dengue 2 virus envelopeglycoprotein in insect cells. A) illustration of relative positions ofdengue 2 virus structural protein genes capsid ©, premembrane (prM) andenvelope (E), and the N-terminal end of the adjacent non-structuralprotein NS1; B) nucleotide coordinates of the E gene construct used forinsertion into shuttle vector pBluBacIII, extending from nucleotides 844to 2422, including a sequence from vector identifying relative positionsof the beta-galactosidase gene (lacZ), polydedrin promoter (Pph),BglII/PstI cloning site, recombination sequences and ampicillinresistance gene (amp).

FIG. 2. Gel filtration of dengue 2 virus recombinant envelopeglycoprotein (rEgp) expressed by baculovirus using a column of G100Sephadex. The column was equilbrated in phosphate buffered saline (PBS)and fractions were eluted in PBS. Fractions were assayed for antigenicreactivity using the antigen dot blot assay and hyperimmune murineascites fluid specific for dengue 2 virus. Data are plotted asabsorbance at 280 nanometers (A260 nm) and counts per minute vesesfraction number. Solid line represents relative absorbance of the sampleat 280 nm; dashed line represents antigenic activity; dotted linerepresents the elution pattern of column calibration standardsthyroglobulin, 670 kilodaltons (kD), bovine gamma globulin, 158 kD,chicken ovalbumin, 44 kD and myoglobin, 17 kD.

FIGS. 3. (A-B) Chromatographic analysis of recombinant dengue 2 virusenvelope glycoprotein (rEgp) expressed by baculovirus using fastpressure liquid chromatography (FPLC) and a Superose 6 column. Thecolumn was equilibrated with phosphate buffered saline (PBS) and proteinwas eluted with the same. A. Column fractions were assayed for antigenusing anti-dengue 2 hyperimmune ascited fluid in a dot blot assay. Dataare plotted as absorbance at 280 nanometers (A260 nm) and counts perminute (y axis) vesus fraction number. B. The column as calibrated withmolecular weights standards thyroglobulin, 670 kilodaltons (kD), bovinegamma globulin, 158 kD, chicken ovalbumin, 44 kD and myoglobin, 17 kD.

FIGS. 4. (A-D) Effect of sarkosyl on chromatographic elution profile ofrecombinant dengue 2 virus envelope glycoprotein (rEgp) analyzed using aSuperose 6 column and fast pressure liquid chromatography (FPLC). Thecolumn was equilibrated in phosphate buffered saline (PBS) containing0.1% sodium sarkosyl and protein containing recombinant dengue 2envelope glycoprotein was eluted in PBS containing: A) 0.1% sarkosyl, B)1.0% sarkosyl, C) 20% sarkosyl and D) 3.0% sarkosyl. Column fractionswere assayed for antigenic activity using anti-dengue 2 hyperimmuneascites fluid in a dot blot assay. Data are plotted as absorbance (solidline) at 280 nanometers (A260) and counts per minute (dotted line) vesusfraction.

FIGS. 5. (A-C) Effect of sonication on chromatographic elution profileof recombinant dengue 2 virus envelope glycoprotein (rEgp) analyzedusisng a Superose 6 column and fast pressure liquid chromatography(FPLC). Insect cells (Trichoplusia ni) infected with recombinantbaculovirus expressing the dengue 2 virus envelope glycoprotein weresonicated in phosphate buffered saline (PBS) for 0, 20 and 30 minutesand eluted from a Superose 6 column by fast pressure liquidchromatography (FPLC). Solid line represents relative amounts of proteindetected by absorbancy at 280 nanometers (A260) and dotted line (countsper minute) represents antigenic reactivity of fraction aliquots withanti-dengue 2 hyperimmune ascites fluid in a dot blot assay.

FIG. 6. Sucrose gradient centrifugation distribution of recombinantdengue 2 virus envelope glycoprotein (rEgp). Insect cells (Spodopterafrugiperda) infected with recombinant baculovirus were pelleted at lowspeed and protein remaining in the supernatant was pelleted at 100,000 xg for 2.5 hours. The resulting microsomal pellet was subjected todensity gradient ultracentrifugation at 100,000 x g for 2.5 hours usinga stp gradient of 5-30% sucrose in phosphate buffered saline (PBS).Fractions were assayed for antigenic activity (shaded area) usinganti-dengue 2 hyperimmune ascites fluid in a dot blot assay.

FIGS. 7. (A-B) Polyacrylamide gelelectrophoresis and immunoblot analysisof baculovirus-expressed dengue 2 virus recombinant envelopeglycoprotein (rEgp). The micorsomal pellet (described, FIG. 6) wasultracentrifuged through a cushion of 30% sucrose in phosphate bufferedsaline (PBS) for 2.5 hours at 100,000 x g. Proteins in the microsomalpellet or 30% sucrose pellet were resuspended in PBS, sonicated brieflyand bolied in SDS sample buffer for 5 minutes before electrophoresis ona 10% SDS polyacrylamide gel. A) Coomassie-blue stained gel: lane 1,molecular weight standard; lane 2, microsomal pellet; lanes 3 and 4, 30%sucrose pellets (contained in 10 or 20 microliters respectively). B)Proteins were electrophoretically transferred to nitrocellulose paperand this immunoblot was probed with hyperimmune mouse ascites fluidspecific for dengue 2 virus. Lanes in B correspond to lanes in A.

DETAILED DESCRIPTION

In one embodiment, the present invention relates to a DNA or cDNAsegment which encodes the complete E protein of DEN-2 and the carboxyterminus of membrane/premembrane protein extending from nucleotide 844to 2422 of the DEN-2 viral genome and including linear andconformational, neutralizing epitopes said sequence identified as SEQ IDNO: 1.

DNA sequences to which the invention also relates include sequenceswhich encode the specific protein epitopes within said sequence whichelicit neutralizing antibody production in animals upon administrationof the protein encoded by said DNA sequences. Specifically, suchsequences include regions encoding neutralizing epitopes present on thenucleotide sequence encompassing amino acids 1 through 495 of the Eprotein several of which have been mapped (Henchel, E. et al. Am. J.Trop. Med. Hyg., 1985, 34: 162-167) and found to be conformational aswell as linear epitopes examples of which are found in TABLE 1 underResults section.

In another embodiment, the present invention relates to a recombinantDNA molecule that includes a vector and a DNA sequence as describedabove (advantageously, a DNA sequence encoding the protein having theneutralizing antibody-eliciting characteristics of that protein). Thevector can take the form of a virus shuttle vector such as, for example,baculovirus vectors pBlueBac-III, pBlueBac-HIS-A-B-C, MaxBac; a plasmid,or eukaryotic expression vectors such as such as GST gene fusionvectors, pGEx-3x, pGEx-2T, pGEx, mammalian cell vectors (pMSG, pMAMneo)or vectors for expression in drosophila or yeast, in addition to othervectors known to people in the art. The DNA sequence can be present inthe vector operably linked to regulatory elements, including, forexample, a promoter or a highly purified human IgG molecule, for exampleProtein A, an adjuvant, a carrier, or an agent for aid in purificationof the antigen as long as the rEgp is expressed as a particle. Therecombinant molecule can be suitable for transforming transfectingeukaryotic cells for example, mammalian cells such as VERO or BHK cells,or insect cells such as Sf-9 (Spodopter frugiperda), C6/36 (Aedesalbopictus), and Trichoplusia ni (High five) mosquito cells, Drosophilacells, and yeast (Ssccharomyces cerevisiae) among others.

In another embodiment, the present invention relates to a recombinantprotein having an amino acid sequence corresponding to SEQ ID NO: 2 andencompassing 495 amino acids of the E protein and 31 amino acids of thecarboxy-terminus of the adjacent M/prM protein from DEN-2 or any allelicvariation thereof which maintains the neutralizing antibody productioncharacteristic of the recombinant protein. As an example, the protein(or polypeptide) can have an amino acid sequence corresponding to anepitope such as a B-cell and T-cell epitope present on the envelopeglycoprotein of DEN-2, or conformational epitopes examples of which arefound in TABLE 1. In addition, the protein or polypeptide, or a portionthereof, can be fused to other proteins or polypeptides which increaseits antigenicity, thereby producing higher titers of neutralizingantibody when used as a vaccine. Examples of such proteins orpolypeptides include any adjuvants or carriers safe for human use, suchas aluminum hydroxide and liposomes.

In yet another embodiment, the present invention relates to arecombinant protein as described above which is capable of assemblinginto more than one protein unit. Assembly of the individual proteinunits can be by hydrophobic forces, or chemical forces, by cross-linkingreagents, or the assembled protein can be further stabilized bycross-linking reagents, and liposomes. The particle can encompass fromat least 2 units of envelope protein. Such a particle can provide higherimmunogenicity and possibly cross-link cell surface immunoglobulins on Bcells.

In a further embodiment, the present invention relates to host cellsstably transformed or transfected with the above-described recombinantDNA constructs. The host cell can be lower eukaryotic (for example,yeast or insect) or higher eukaryotic (for example, all mammals,including but not limited to mouse and human). For instance, transientor stable transfections can be accomplished into CHO or Vero cells.Transformation or transfection can be accomplished using protocols andmaterials well known in the art. The transformed or transfected hostcells can be used as a source of the DNA sequences described above. Whenthe recombinant molecule takes the form of an expression system, thetransformed or transfected cells can be used as a source of theabove-described recombinant protein.

In a further embodiment, the present invention relates to a method ofproducing the recombinant protein which includes culturing theabove-described host cells, under conditions such that the DNA fragmentis expressed and the recombinant protein is produced thereby. Therecombinant protein can then be isolated using methodology well known inthe art. The recombinant protein can be used as a vaccine for immunityagainst infection with flaviviruses or as a diagnostic tool fordetection of viral infection.

In yet another embodiment, the present invention relates to a method ofpurifying the recombinant protein particles, said method comprising thesteps of:

(i) harvesting cells expressing recombinant DEN envelope glycoprotein;

(ii) separating a cell pellet and a supernatant from said harvestedcells;

(iii) lysing said cell pellet of step (ii) to release recombinantenvelope glycoprotein;

(iv) pelleting said recombinant envelope glycoprotein from said lysedcells;

(v) fractionating said recombinant envelope glycoprotein from steps (ii)and (v) through a density gradient;

(vi) collecting purified recombinant envelope glycoprotein from pellet.

The density gradient of step (vi) may be made of any density separationmaterial such as cesium chloride, ficoll, or molecular sieve material.The recombinant envelope glycoprotein can also be pelleted from saidsupernatant. If desired, the cell debris can be pelleted or separatedfrom said recombinant envelope glycoprotein after lysing cell pellet asdescribed in (iii).

In a further embodiment, the present invention relates to a method ofdetecting the presence of DEN virus disease or antibodies against DENvirus in a sample. Using standard methodology well known in the art, adiagnostic assay can be constructed by coating on a surface (i.e. asolid support) for example, a microtitration plate or a membrane (e.g.nitrocellulose membrane), all or a unique portion of the recombinantenvelope protein particle described above, and contacting it with theserum of a person suspected of having DEN fever. The presence of aresulting complex formed between the recombinant protein and antibodiesspecific therefor in the serum can be detected by any of the knownmethods common in the art, such as fluorescent antibody spectroscopy orcolorimetry. This method of detection can be used, for example, for thediagnosis of DEN disease. This method when employing distinct rEgpparticles specific for each DEN serotype, will allow the detection ofthe presence of each respective DEN serotype in a sample. Infection withmore than one serotype is thought to play a role in the etiology of DENhaemorrhagic fever and DEN shock syndrome.

In addition, the present invention is related to a method of detectingflavivirus disease or antibodies against flavivirus in a sample. Dengueviruses are members of the family Flaviridae which includes over sixtymembers among which there is considerable genetic and antigenicsimilarity but no significant cross-neutralization. It would be apparentto persons in the art to apply the concepts of the present inventionexemplified in DEN-2 to similar proteins and DNA sequences present inother related flaviviruses such as yellow fever, Japanese encephalitisand tick-borne encephalitis viruses.

In another embodiment, the present invention relates to a diagnostic kitwhich contains the recombinant envelope protein particle and ancillaryreagents that are well known in the art and that are suitable for use indetecting the presence of antibodies to flavivirus antigens in serum ora tissue sample, specifically antibodies to DEN virus. Tissue samplescontemplated can be monkey and human, or other mammals.

In another embodiment, the present invention relates to a vaccine forprotection against a flavivirus disease. The vaccine can be prepared byinducing expression of the recombinant expression vector described abovein either a higher mammalian or lower (insect, yeast, fungi) eukaryotichost and purifying the recombinant glycoprotein particle describedabove. The purified particles are prepared for administration to mammalsby methods known in the art, which can include preparing the particleunder sterile conditions and adding an adjuvant. The vaccine can belyophilized to produce a flavivirus vaccine in a dried form for ease intransportation and storage. Further, the vaccine may be prepared in theform of a mixed vaccine which contains the recombinant protein describedabove and at least one other antigen as long as the added antigen doesnot interfere with the effectiveness of the dengue vaccine and the sideeffects and adverse reactions are not increased additively orsynergistically. It is envisioned that a tetravalent vaccine composed ofrecombinant antigenic proteins from the four serotypes of dengue virus,DEN-1, DEN-2, DEN-3, and DEN-4 can be produced to provide protectionagainst dengue disease.

The vaccine may be stored in a sealed vial, ampoule or the like. Thepresent vaccine can generally be administered in the form of a liquid orsuspension. In the case where the vaccine is in a dried form, thevaccine is dissolved or suspended in sterilized distilled water beforeadministration. Generally, the vaccine may be administeredsubcutaneously, intradermally or intramuscularly in a dose effective forthe production of neutralizing antibody and protection from infection.

In another embodiment, the present invention relates to a naked DNA orRNA vaccine. The DEN DNA fragment, of the present invention described inSEQ ID NO: 1 or a portion thereof, or an allelic form thereof, can beadministered as a vaccine to protect against DEN virus disease and toelicit neutralizing antibodies against the virus. The DNA can beconverted to RNA for example by subcloning the said DNA into atranscriptional vector, such as pGEM family of plasmid vectors, or undercontrol of a transcriptional promoter of a virus such as vaccinia, andthe RNA used as a naked RNA vaccine. It is understood and apparent to aperson with ordinary skill in the art that due to the similarity betweendifferent serotypes of DEN as well as similarities between flaviviruses,a DNA sequence from any DEN serotype or flavivirus encoding the completeenvelope protein of its respective flavivirus can be used as a naked DNAvaccine against infection with its respective virus. The DEN-2 naked DNAor RNA vaccine can be injected alone, or combined with at least oneother antigen or DNA or RNA fragment as long as the added antigen or DNAor RNA fragment does not interfere with the effectiveness of the DENvaccine and the side effects and adverse reactions are not increasedadditively or synergistically. It is envisioned that a tetravalentvaccine composed of DNA or RNA fragments from the four serotypes ofdengue virus, DEN-1, DEN-2, DEN-3, and DEN-4 can be produced to provideprotection against dengue disease.

The naked DNA or RNA vaccine of the present invention can beadministered for example intermuscularly, or alternatively, can be usedin nose drops. The DNA or RNA fragment or a portion thereof can beinjected as naked DNA or RNA, as DNA or RNA encapsulated in liposomes,as DNA or RNA entrapped in proteoliposomes containing viral envelopereceptor proteins (Nicolau, C. et al. Proc. Natl. Acad. Sci. U.S.A.1983, 80, 1068; Kanoda, Y., et al. Science 1989, 243, 375; Mannino, R.J. et al. Biotechniques 1988, 6, 682). Alternatively, the DNA can beinjected along with a carrier. A carrier can be a protein or such as acytokine, for example interleukin 2, or a polylysine-glycoproteincarrier (Wu, G. Y. and Wu, C. H. J. Biol. Chem. 1988, 263, 14621), or anonreplicating vector, for example expression vectors containing eitherthe Rous sarcoma virus or cytomegalovirus promoters. Such carrierproteins and vectors and methods for using same are known to a person inthe art (See for example, Acsadi, G. et al. Nature 1991, 352, 815-818).In addition, the DNA or RNA could be coated onto tiny gold beads andsaid beads introduced into the skin with, for example, a gene gun(Cohen, J. Science 1993, 259, 1691-1692; Ulmer, J. B. et al. Science1993, 259, 1745-1749).

Described below are examples of the present invention which are providedonly for illustrative purposes, and not to limit the scope of thepresent invention. In light of the present disclosure, numerousembodiment within the scope of the claims will be apparent to those ofordinary skill in the art.

The following MATERIALS AND METHODS were used in the examples thatfollow.

Cells and viruses. Dengue-2 virus was propagated in Aedes albopictuscells (C6/36 cells, American Type Tissue Culture Collection, ATCC,Rockville, Md.). To propagate virus, C6/36 cells were grown at 28° C. inCO₂ -independent medium (Gibco, Grand Island, N.Y.) containing 10% fetalbovine serum (FBS, heat inactivated at 56° C. for 30 min, Sigma, StLouis, Mo.). Wild-type DEN-2 virus (strain PR 159) was the source ofgenomic RNA for synthesis of the rEgp gene. A mouse-adapted New Guinea Cstrain was used for immunizations and plaque neutralization assays.African green monkey kidney cells were purchased from ATCC. Baculovirus(Autographa californica nuclear polyhedrosis virus, AcPNV, Invitrogen,San Diego, Calif.) was propagated in Spodoptera frugiperda (Sf-9 andSf-21) and Trichoplusia ni (High five) cells (Invitrogen). High fivecells and Sf-9 cells were cultured in tissue culture flasks at 28° C. inTNMFH medium (Biowhittaker, Walkersville, Md.) supplemented with 10%FBS, penicillin (100 units U/ml), streptomycin (100 μg/ml), glutamine (2mM) and gentamycin (50 mg/ml). Recombinant baculoviruses were isolatedin Sf-9 cells following previously described procedures (5). The Sf-21cells were grown in 10 liter spinner culture in TNMFH media supplementedas above for High five and Sf-9 cells.

Cloning of the DEN-2 envelope gene. The gene encoding the DEN-2 Egp andan adjacent upstream translocation signal sequence (Markoff, L., J.Virol., 1989, 63:3345-3352) was derived by reverse transcription ofviral genomic RNA followed by amplification of cDNA by the polymerasechain reaction. Dengue-2 virus RNA was purified from supernatants ofvirus-infected C6/36 cells by guanidine isothiocyanatephenolchloroform:isoamyl alcohol extraction (Chomczynski and Sacchi, AnalBiochem., 1987, 162:156-159). Primers were constructed that incorporatedenzyme restriction sequences onto ends of the Egp gene fragment, and thefragment was inserted into the baculovirus transfer vector pBlueBacIII(Invitrogen). The sequence of the recombinant Egp (rEgp) gene fragmentin pBlueBacIII was determined to be identical to that of the native Egpgene (Hahn, et al. Virology, 1988, 185:401-410) by dideoxy sequencing(Sanger et al., Proc. Nail. Acad. Sci. U.S.A., 1977, 74: 5463-5467).

Cotransfection and purification of recombinant baculoviruses.Recombinant baculoviruses were generated by co-transfecting Sf-9 cellswith a recombinant pBlueBac III plasmid together withcommercially-prepared linear baculovirus (Invitrogen, San Diego,Calif.). The Egp gene fragment was transferred into the baculovirusgenome by homologous recombination (Summers and Smith, A Manual ofMethods for Baculovirus Vectors and Insect Cell Culture Procedure. TexasAgricultural Experimental Station Bulletin No. 1555, Texas AgriculturalStation, College Station, Tex., 1987). Plaque assays in Sf-9 cells wereused to isolate the recombinant baculovirus clones which yielded blueplaques due to the transfer of the β-galactosidase gene from thepBlueBac III plasmid. Following infection of Sf-9 cells with a plaquepurified recombinant baculovirus clone, DNA was extracted from cells andthe presence of the Egp gene was confirmed by hybridization of a ³²P-labeled Egp gene probe with the DNA.

SDS-polyacrylamide gel electrophoresis and western blotting. Proteinswere resolved on a 10% SDS-polyacrylamide gel (Laemmli, U. K. Nature,1970, 227:680-685). Samples were either boiled for 5 minutes or notboiled before application to the gel. Proteins were blotted ontonitrocellulose paper using a dry blot apparatus (Enprotech, IntegratedSeparation Systems, Hyde Park, Mass.) as recommended by themanufacturer. Following protein transfer, the nitrocellulose was blockedfor 30 minutes in PBS-0.05% azide containing 5% powdered milk (blockingbuffer) and incubated overnight in blocking buffer containing a 1:500dilution of anti-DEN-2 hyperimmune mouse ascites fluid (HMAF, 11). Theblot was washed 3 times in PBS containing 0.05% Tween 20 (PBS-T) andincubated for 1 hour in alkaline phosphatase-conjugated goat anti-mouseIgG (Kirkegaard and Perry, Gaithersburg, Md.). The blot was washed 3times in PBS-T and finally in Tris-glycine-saline, (TGS), pH 8.0.Antigenic bands were visualized by incubating the blot in TGS containing2 mg/ml napthol and 1 mg/ml phenol red (Sigma, St Louis, Mo.).

Antigen dot blot. Samples were applied to nitrocellulose paper using a96-well manifold under vacuum. The paper was blocked and incubatedovernight in blocking buffer containing HMAF diluted 1:500. The paperwas washed 3 times with PBS-T and incubated for 1 hour in blockingbuffer containing goat anti-mouse immunoglobulin gamma (Kirkegaard andPerry) labeled with ¹²⁵ I (Gentry M. K. et al., Am. J. Trop. Med. Hyg.,1982, 31: 548-555), using labeled antibody at 10⁶ cpm/ml of blockingbuffer. Following incubation with the labeled antibody, the paper waswashed 3 times with PBS-T, cut into sample squares and counted in aclinical gamma counter (Pharmacia-LKB, Piscataway, N.J.).

Antibody Affinity Assays. A particle fluorescence assay (PFCIA) wasdeveloped based on previous methodologies (Scatchard, G. Ann. N.Y. Acad.Sci., 1989, 51:660-672; Schots et al. Virology, 1988, 162:167-180) toquantitate fluorescence in an antibody-antigen binding assay usingFITC-labeled purified mAbs. The amount of fluorescence, via antibody,bound to antigen adsorbed to polystyrene beads was assayed usingpolycarbonate IDEXX assay plates (IDEXX, Westbrook, Me.) and a PFCIAanalyzer (IDEXX). Binding affinities of the three mAbs were measuredunder neutral (pH 7.0) and acidic (pH 5.0) buffering conditions.Antigens tested in the assay were: rEgp derived from the cell lysatedescribed above, partially purified rEgp obtained by columnfractionation (see below) of the cell lysates, or DEN-2 virus (NGCstrain). Antigen and serially-diluted FITC-conjugated Egp-specific mAbs(100 μg/ml) were separately adsorbed onto polystyrene beads (IDEXX) for1 hour at room temperature. Protein-bound beads were washed twice in PBSand resuspended in PBS containing 0.1% bovine serum albumin (BSA) and0.1% sodium azide at a final particle concentration of 0.25% w/v. Theassay was conducted in triplicate for each mAb dilution. For the assay,blocking buffer (PBS containing 1% BSA) was distributed into wells ofIDEXX plates followed by the addition of antigen-coated beads. Serialdilutions of FITC-labeled mAbs were then added to the wells and plateswere incubated in the analyzer for washing (PBS, pH 7.2, 0.1% BSA and0.02% Tween) and fluorescence quantitation. Results were analyzed by theLigand software program, PJ Munson, Division of Computer Research andTechnology, The National Institutes of Health, Bethesda, Md.

Gel Filtration Chromatography. Clarified supernatants of lysed, infectedHigh five cells were strained through a 0.4 micron filter andfractionated by gravity flow using a column of Sephadex G-100 (1.5×30cm) or by Fast Pressure Liquid Chromatography (Pharmacia) using columnsof Sepharose-6 and Sepharose 12 (2.5×60 cm). Fractions were collectedand aliquots of the fractions were assayed for antigenic activity byantigen dot blot assay.

Purification of rEgp by ultracentrifugation. Infected High five or Sf-21cells were harvested, pelleted by low-speed centrifugation and washedseveral times with PBS. The pellet was disrupted by sonication andclarified by low-speed centrifugation. The supernatant was centrifugedat 100,000×g for 90 minutes, and the microsomal pellet was collected.The pellet was sonicated and centrifuged at 100,000×g for 3 hoursthrough either a step gradient of 5 to 30% sucrose in PBS, or through a30% sucrose cushion. Fractions collected were dialyzed against PBSbefore testing.

Mouse immunizations and challenge. Groups of ten, 4-6-week old femaleBALB/c mice (Jackson Laboratories, Bar Harbor, Me.) were immunizedsubcutaneously with doses of 0.4, 1.0 and 4.0 μg of purified rEgp in 0.5ml without adjuvant or with antigen adsorbed onto Alhydrogel (Alum,Superfos Biosector, Denmark). A control group of 10 mice was immunizedwith either PBS or 10⁴ plaque forming units (pfu) of DEN-2 virus (NGCstrain). After 28 days, animals were boosted once with antigen, PBS orvirus. Two weeks following the boost, half of the mice of each groupwere bled and individual sera were tested in plaque reductionneutralization assays. The other half of the mice of each group werechallenged intracerebrally with 10⁴ pfu of DEN-2 virus (NGC strain).After 5 days, mice were sacrificed, brains were aseptically removed,homogenized and used in a plaque assay to quantitate viral growth.

Plaque reduction neutralization test (PRNT) and viral plaque assay. Micewere immunized on days 0 and 30 and bled 2 weeks following the boost.Sera collected from immunized mice at days were serially dilutedten-fold and incubated at 37° for 1 hour with 250 pfu/ml of DEN-2 virus(NGC strain). Following incubation, 2 ml aliquots of the sera-virusmixture was distributed onto duplicate monolayers of Vero cells in6-well plates. After plates were rocked for 1 hour at 37° C., monolayersan overlay of 1% melted agarose in 2× EMEM was added onto eachmonolayer. After 6 days of incubation at 37° C., a second overlay ofagarose containing a neutral red stain was applied, and plates wereincubated overnight at 37° C. Viral plaques were counted the followingday.

To quantitate viral growth, brain tissue homogenates serially dilutedten-fold were distributed onto Vero cell monolayers and incubated asdescribed above. A garose overlays were added and viral plaques werecounted as describe above.

RESULTS

Construction of recombinant pBlueBacIII transfer vector. The DEN-2 Egpgene fragment that was inserted into pBlueBacIII shown in FIG. 1. Thefragment encodes the full Egp (495 amino acids) and 31 amino acids ofthe C terminus of the adjacent upstream M/preM protein. This segmentserves as a signal for membrane translocation of the Egp (Markoff, L. J.Virol. 1989, 63:3345-3352)). Synthetic primers used to amplify the genefragment each contained 18 nucleotides complementary to specificsequences in the DEN-2 E gene. The forward primer contains a Bgl IIenzyme restriction site and an ATG start codon (SEQ ID NO:3). Thereverse primer contains a Pst I enzyme restriction site and a stopcodon. The E gene fragment was cut with Bgl II and Pst I enzymes andinserted unidirectionally into the BglII-Pst I cloning site of thepBlueBac III plasmid placing the recombinant gene was under the controlof the AcNPV polyhedrin promoter.

Antigenicity of baculovirus-vectored rEgp. To perform an epitopeanalysis of the rEgp, the protein suspension containing rEgp andpurified DEN-2 virus were reacted in an antigen dot blot assay with apanel of mAbs. The panel contained mAbs that bind either linear ordiscontinuous antigenic sites, and recognize both neutralizing andnon-neutralizing epitopes. Results of the assay showed that the rEgpreacted to every mAb in the panel (Table 1). Since reactivities by thisassay were quantitatively different for individual epitopes, bindingaffinities of the individual mAbs to the rEgp and native Egp weredetermined. The mAbs selected for affinity assays, 2H3, 4G2, and 9D12,demonstrated weak (2H3) to strong (9D12) binding to the rEgp in theantigen dot blot assay. Table 2 shows that the binding affinities ofindividual mAbs for rEgp and partially purified rEgp was comparable totheir affinities for virus. Binding assays conducted at both neutral andslightly acidic pH demonstrated that these epitopes were not affected bypH.

                  TABLE 1                                                         ______________________________________                                        Antibody binding of the dengue-2 recombinant envelope protein                   expressed by baculovirus                                                            Reactivity with antigen.sup.b                                         Antibody.sup.a                                                                        ACNPV-E   ACNPV-prME DEN-2 Virus                                                                            ACNPV                                   ______________________________________                                        3H5.sup.d                                                                              13.6.sup.c                                                                             10.1       7.5      1.4                                       9D12.sup.d, e 12.3 12.1  9.0 1.0                                              13B7 10.5 4.1 5.6 3.6                                                         4E5.sup.d  8.6 6.6 10.5  1.0                                                  2H3.sup.d  4.9 2.5 11.5  1.9                                                  4G2.sup.d, e  8.5 5.0 16.8  1.0                                               1B7.sup.d, e  5.1 3.1 8.9 1.2                                                 HMAF 13.5 17.6  7.0 1.2                                                       HCS 12.5 NT 12.5  1.6                                                       ______________________________________                                         .sup.a Antibodies were diluted 1:100 (mAbs) or 1:500 (antiDEN-2               hyperimmune mouse ascites fluid, HMAF; or convalescent human sera, HCS).      .sup.b Antigenicity reactivity of extracts from High5 cells infected with     recombinant baculovirus clones containing DEN2E (ACNPVE) or prME              (ACNPVprME) genes, tested by antigen dot blot assay. Purified DEN2 virus      served as the positive control in the assay. Protein extracted from High5     cells infected with wildtype baculovirus served as the negative control.      .sup.c Antigenantibody biding was detected by .sup.125 Ilabeled goat          antimouse immunoglobulin. Data for each mAb and HMAF represents an averag     of three separate experiments; and for HCS, one experiment. Results are       give as cpm × 10.sup.3.                                                 .sup.d Antibodies which neutralize virus infectivity in vitro (Henchal et     al. Am J. Trop. Med. Hyg. 1985, 34:162-167).                                  .sup.e Antibodies which recognize conformational epitopes (Henchal et al.     Am J. Trop. Med. Hyg. 1985, 34:162-167; Megret et al. Virology, 1992,         187:480-491).                                                            

                  TABLE 2                                                         ______________________________________                                        Binding affinity of monoclonal antibodies to recombinant and native            dengue-2 envelope proteins                                                    Affinity binding of mAbs 9D12, 2H3, and 4G2 at pH 5.0                            Antigen.sup.a                                                                          9D12          2H3    4G2                                         ______________________________________                                        Purified 0.4 × 10.sup.-6                                                                       3.2 × 10.sup.-6                                                                  2.3 × 10.sup.-6                           Lysate 0.5 × 10.sup.-6 1.0 × 10.sup.-6 2.9 × 10.sup.-6      Virus 5.2 × 10.sup.-6 2.0 × 10.sup.-6 1.3 × 10.sup.-6     ______________________________________                                         .sup.a Antigens were either partiallypurified recombinant E protein,          lysates of cells infected with the Eprotein recombinant baculovirus, or       purified DEN2 virus.                                                     

Analysis of the antigenic properties of the full DEN-2 rEgp expressed inthis study by baculovirus demonstrated that properly conformed proteinscan be produced in this system. This was evidenced by the strongreactivity of the rEgp with mAbs that represented both linear andconformational-dependent epitopes within the native protein. Bindingaffinities of selected mAbs to native epitopes were not modified in therecombinant protein.

The mAb binding assays qualitatively demonstrate that native proteinepitopes were preserved on the recombinant E protein.

Gel filtration analysis of DEN-2 rEgp particles. The DEN-2 Egp wasexpressed from baculovirus in High-five and Sf-21 cells. Cells werelysed by sonication in PBS containing 0.1% sarkosyl. Gel filtration ofthe cell lysates shows that the majority of rEgp produced by baculovirushad self-aggregated to form high molecular weight particles. Proteinseparation profiles for infected cell lysates are shown in FIGS. 2, 3, 4and 5. Antigenic reactivity with anti-DEN-2 HMAF is distributed amongnearly all fractions passed through G-100 Sephadex, with a majorantigenic peak eluting at the position of calibration standardthyroglobulin, molecular weight (mol wt) 670 kilodaltons (kd). Similarresults were obtained for gel FPLC using Superose 6 (FIG. 3) andSuperose 12 (FIGS. 4 and 5). The rEgp was eluted in the void volume ofthe Superose 6 column (molecular weight exclusion, 5×10⁶ kd) infractions 8 through 11 compared to the calibration standardthyroglobulin which was eluted in fractions 13 and 14. Similarly, therEgp eluted in the void volume of the Superose 12 column (mol wtexclusion, 3×10⁵).

The role of sarkosyl and sonication in disruption of rEgp particles wasalso examined during Superose 12 chromatography. By equilibrating columnin increasing amounts of sarkosyl (0.1 to 3.0%), protein elutionprofiles were shifted, however position major antigenic peak associatedwith rEgp was not altered by sarkosyl (FIGS. 4A, B, C, D). Sonicationfor up to 30 minutes did, however, partially disrupt rEgp aggregates aswell as other high molecular weight protein aggregates (FIGS. 5A, B, andC).

Purification of E particles. Gel filtration results indicated that rEgpaggregates could be separated from the majority of cellular proteinsbased on their large size. However, yield of partially purified rEgpproduced in this manner were relatively low and the process was slowedby frequent necessity to clean the column matrix. Aggregated rEgpparticles were therefore purified from other cellular components bydifferential centrifugation using a sucrose cushion. In initialexperiments, the microsomal fraction of infected cell lysates wascollected by ultracentrifugation, sonicated, and centrifuged through a5-30% sucrose step gradient. Fractions containing 500 μl wereconcentrated, dialyzed and analyzed for reactivity with anti DEN-2 mouseHMAF. FIG. 6 shows that very high antigenic activity was present in thegradient pellet, compared to relatively small amount of antigenicactivity that was distributed into several gradient fractions. Since themajority of E antigen was present in the 5-30% sucrose gradient pellet,E protein aggregates were purified by centrifugation of the microsomalfraction through a 30% sucrose cushion.

SDS-polyacrylamide gel electrophoresis and western blotting. Proteinsthat were pelleted through the 30% sucrose cushion were analyzed bySDS-PAGE and western blotting. As shown in FIG. 7A, this pelletcontained three protein bands that stained with Coomassie blue on a 10%reduced SDS-polyacrylamide gel. A western blot of a non-reduced 10% gelloaded with identical samples revealed three antigenic bands that appearto correspond to the three protein-stained bands (FIG. 7B). These bandsseen on the protein gel and on the blot migrate close together, and arelikely to represent varying degrees of glycosylation of the rEgp.

Immunogenicity of the purified rEgp. The purified rEgp particles weretested in immunogenicity trials in mice. Previously it was shown that acellular lysate containing baculovirus vectored rEgp was fully reactivewith native E-specific monoclonal antibodies and induced a low titer ofneutralizing antibody in mice. Table 3 shows results from immunizationof mice with purified rEgp. Mice responded to immunization by productionof neutralizing antibodies. Table 3 shows that a non-adjuvantedimmunizing dose of 4 μg induced production of neutralization antibodies.This response was boosted several fold when rEgp was pre-adsorbed toAlum, and was equivalent to titers induced by live virus. Thepre-absorption to alum also increased the response with 1 μg to adetectable level (Table 3).

                  TABLE 3                                                         ______________________________________                                        PRNT.sub.50  in mice immunized with baculovirus expressed DEN-2 E protein       rEgp with Alum                                                                              rEgp/no adjuvant                                              Dose 4 μg                                                                         Dose 1 μg                                                                           Dose 4 μg                                                                           Dose 1 μg                                                                         DEN-2 NGC                                                                             Control                               ______________________________________                                        >850   458      472      <13    633     <13                                     >850 280 233 <13 526 <13                                                       441 473 538 <13 480                                                          >850 476 261 <13 622                                                           540 788  23 ND 574                                                         ______________________________________                                         *Vaccination schedule: Day 1 and day 30. Bled two weeks after the second      dose.                                                                    

Mice immunized with purified non-adjuvanted and adjuvanted rEgp weretested in a challenge assay with live virus. Table 4 shows results forgrowth of DEN-2 challenge virus in immunized and control mice.

                  TABLE 4                                                         ______________________________________                                        Percent protection measured by reduction of dengue virus in the brains of      immunized, intracerebrally challenged mice                                         Immunization.sup.1                                                                          Percent reduction.sup.2                                   ______________________________________                                        4 μg with alum                                                                             88.5 ± 35.1                                                  1 μg with alum  97 ± 4.2                                                0.25 μg with alum   92.6 ± 5.1                                            4 μg without alum 83.4 ± 40.2                                           1 μg without alum 78.5 ± 24.7                                         0.25 μg without alum 96.8 ± 3.8                                         live virus 100 ± 0                                                         none  0 ± 86                                                             ______________________________________                                         .sup.1 Mice were immunized at days 0 and 30 with indicated amounts of         recombinant dengue 2 envelope protein or with live dengue 2 virus. Contro     mice were not immunized.                                                      .sup.2 Mice were inoculated intracerebrally with 10,000 pfu of mouse          adapted dengue 2 virus two weeks after the last immunization. Five days       later, mice were euthanized and the brains removed for quantitation of        dengue virus in the brain. The percent reduction was calculated by            multiplying 100 times the formula (controlplagues/control) where control      is the mean of the virus plaques in un immunized mice and plaques is  #th     plaques measured in individual mice. Results are displayed as the mean        ± the standard deviation for the mice in each group.                  

Table 4 shows that the mean number of viral plaques obtained from brainsof all groups of immunized mice were greatly reduced compared to thatobtained in unimmunized mice. Mean number of plaques obtained for miceimmunized with adjuvanted antigen (groups 1, 2 and 3 mice, immunizedwith 4, 1, and 0.4 μg of rEgp, respectively), were significantly lowerthan those obtained for mice immunized with non-adjuvanted rEgp.

    __________________________________________________________________________    #             SEQUENCE LISTING                                                   - -  - - (1) GENERAL INFORMATION:                                             - -    (iii) NUMBER OF SEQUENCES:  4                                          - -  - - (2) INFORMATION FOR SEQ ID NO:1:                                     - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 1578 base - #pairs                                                (B) TYPE: Nucleic acid                                                        (C) STRANDEDNESS: Single                                                      (D) TOPOLOGY: Linear                                                 - -     (ii) SEQUENCE DESCRIPTION: SEQ ID NO:1:                               - - ATGGCCGCAA TCCTGGCATA CACCATAGGA ACGACGCATT TCCAAAGAGT CC -            #TGATATTC     60                                                                 - - ATCCTACTGA CAGCCATCGC TCCTTCAATG ACAATGCGCT GCATAGGAAT AT -            #CAAATA      120                                                                 - - GACTTTGTGG AAGGAGTGTC AGGAGGGAGT TGGGTTGACA TAGTTTTAGA AC -            #ATGGAAG     180                                                                 - - TGTGTGACGA CGATGGCAAA AAATAAACCA ACACTGGACT TTGAACTGAT AA -            #AAACAGA     240                                                                 - - GCCAAACAAC CCGCCACCTT AAGGAAGTAC TGTATAGAGG CTAAACTGAC CA -            #ACACGAC     300                                                                 - - ACAGACTCGC GCTGCCCAAC ACAAGGGGAA CCCACCCTGA ATGAAGAGCA GG -            #ACAAAAG     360                                                                 - - TTTGTCTGCA AACATTCCAT GGTAGACAGA GGATGGGGAA ATGGATGTGG AT -            #TATTTGG     420                                                                 - - AAAGGAGGCA TCGTGACCTG TGCCATGTTC ACATGCAAAA AGAACATGGA GG -            #GAAAAAT     480                                                                 - - GTGCAGCCAG AAAACCTGGA ATACACTGTC GTTATAACAC CTCATTCAGG GG -            #AAGAACA     540                                                                 - - GCAGTCGGAA ATGACACAGG AAAACATGGT AAAGAAGTCA AGATAACACC AC -            #AGAGCTC     600                                                                 - - ATCACAGAGG CGGAACTGAC AGGCTATGGC ACTGTTACGA TGGAGTGCTC TC -            #CAAGAAC     660                                                                 - - GGCCTCGACT TCAATGAGAT GGTGTTGCTG CAAATGAAAG ACAAAGCTTG GC -            #TGGTGCA     720                                                                 - - AGACAATGGT TCCTAGACCT ACCGTTGCCA TGGCTGCCCG GAGCAGACAC AC -            #AAGGATC     780                                                                 - - AATTGGATAC AGAAAGAGAC ACTGGTCACC TTCAAAAATC CCCATGCGAA AA -            #AACAGGA     840                                                                 - - GTTGTTGTCT TAGGATCCCA AGAGGGGGCC ATGCATACAG CACTCACAGG GG -            #CTACGGA     900                                                                 - - ATCCAGATGT CATCAGGAAA CCTGCTGTTC ACAGGACATC TTAAGTGCAG GC -            #TGAGAAT     960                                                                 - - GACAAATTAC AACTTAAAGG GATGTCATAC TCCATGTGCA CAGGAAAGTT TA -            #AAGTTG     1020                                                                 - - AAGGAAATAG CAGAAACACA ACATGGAACA ATAGTCATTA GAGTACAATA TG -            #AAGGAG     1080                                                                 - - GGCTCTCCAT GCAAGACCCC TTTTGAGATA ATGGATCTGG AAAAAAGACA TG -            #TTTTGG     1140                                                                 - - CGCCTGACCA CAGTCAACCC AATTGTAACA GAAAAGGACA GTCCAGTCAA CA -            #TAGAAG     1200                                                                 - - GAACCTCCAT TCGGAGACAG CTACATCATC ATAGGAGTGG AACCAGGACA AT -            #TGAAGCT    1260                                                                 - - GACTGGTTCA AGAAAGGAAG TTCCATCGGC CAAATGTTTG AGACAACAAT GA -            #GGGGAG     1320                                                                 - - AAAAGAATGG CCATTTTGGG CGACACAGCC TGGGATTTTG GATCTCTGGG AG -            #GAGTGT     1380                                                                 - - ACATCAATAG GAAAGGCTCT CCACCAGGTT TTTGGAGCAA TCTACGGGGC TG -            #CTTTCA     1440                                                                 - - GGGGTCTCAT GGACTATGAA GATCCTCATA GGAGTTATCA TCACATGGAT AG -            #GAATGA     1500                                                                 - - TCACGTAGCA CATCACTGTC TGTGTCACTG GTATTAGTGG GAATCGTGAC AC -            #TGTACT     1560                                                                 - - GGAGTTATGG TGCAGGCC             - #                  - #                      - #1578                                                                  - -  - - (2) INFORMATION FOR SEQ ID NO:2:                                     - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 526 amino - #acids                                                (B) TYPE: amino acid                                                          (C) TOPOLOGY: Linear                                                 - -     (ii) SEQUENCE DESCRIPTION: SEQ ID NO: - #2:                           - - Met Ala Ala Ile Leu Ala Tyr Thr Ile Gly Th - #r Thr His Phe Gly Arg       1               5  - #                10  - #                15               - - Val Leu Ile Phe Ile Leu Leu Thr Ala Ile Al - #a Pro Ser Met Thr Met                  20      - #            25      - #            30                   - - Arg Cys Ile Gly Ile Ser Asn Arg Asp Phe Va - #l Glu Gly Tyr Ser Gly              35          - #        40          - #        45                       - - Gly Ser Trp Val Asp Ile Tyr Leu Glu His Gl - #y Ser Cys Val Thr Thr          50              - #     55             - #     60                          - - Met Ala Lys Asn Lys Pro Thr Leu Asp Phe Gl - #u Leu Ile Lys Thr Glu      65                  - #70                  - #75                  - #80        - - Ala Lys Gln Pro Ala Thr Leu Arg Lys Tyr Cy - #s Ile Glu Ala Lys Leu                      85  - #                90  - #                95               - - Thr Asn Thr Thr Thr Asp Ser Arg Cys Pro Th - #r Gln Gly Glu Pro Thr                  100      - #           105      - #           110                  - - Leu Asn Glu Glu Gln Asp Lys Arg Phe Val Cy - #s Lys His Ser Met Val              115          - #       120          - #       125                      - - Asp Arg Gly Trp Gly Asn Gly Cys Gly Leu Ph - #e Gly Lys Gly Gly Ile          130              - #   135              - #   140                          - - Val Thr Cys Ala Met Phe Thr Cys Lys Lys As - #n Met Glu Gly Lys Ile      145                 1 - #50                 1 - #55                 1 -      #60                                                                              - - Val Gln Pro Glu Asn Leu Glu Tyr Thr Val Va - #l Ile Thr Pro His        Ser                                                                                             165  - #               170  - #               175             - - Gly Glu Glu His Ala Val Gly Asn Gln Thr Gl - #y Lys His Gln Lys Glu                  180      - #           185      - #           190                  - - Val Lys Ile Thr Pro Gln Ser Ser Ile Thr Gl - #u Ala Glu Leu Thr Gly              195          - #       200          - #       205                      - - Tyr Gly Thr Val Thr Met Glu Cys Ser Pro Ar - #g Thr Gly Leu Asp Phe          210              - #   215              - #   220                          - - Asn Glu Met Val Leu Leu Asp Met Lys Asp Ly - #s Ala Trp Leu Tyr His      225                 2 - #30                 2 - #35                 2 -      #40                                                                              - - Arg Gln Trp Phe Leu Asp Leu Pro Leu Pro Tr - #p Leu Pro Gly Ala        Asp                                                                                             245  - #               250  - #               255             - - Thr Gln Gly Ser Asn Trp Ile Gln Lys Glu Th - #r Leu Val Thr Phe Lys                  260      - #           265      - #           270                  - - Asn Pro His Ala Lys Lys Gln Asp Val Val Va - #l Leu Gly Ser Gln Glu              275          - #       280          - #       285                      - - Gly Ala Met His Thr Ala Leu Thr Gly Ala Th - #r Glu Ile Gln Met Ser          290              - #   295              - #   300                          - - Ser Gly Asn Leu Leu Phe Thr Gly His Leu Ly - #s Cys Arg Leu Arg Met      305                 3 - #10                 3 - #15                 3 -      #20                                                                              - - Asp Lys Leu Gln Leu Lys Gly Met Ser Tyr Se - #r Met Cys Thr Gly        Lys                                                                                             325  - #               330  - #               335             - - Phe Lys Val Val Lys Glu Ile Ala Glu Thr Gl - #n His Gly Thr Ile Val                  340      - #           345      - #           350                  - - Ile Arg Val Gln Tyr Glu Gly Asp Gly Ser Pr - #o Cys Lys Thr Pro Phe              355          - #       360          - #       365                      - - Glu Ile Met Asp Leu Glu Lys Arg His Val Le - #u Gly Arg Leu Thr Thr          370              - #   375              - #   380                          - - Val Asn Pro Ile Val Thr Glu Lys Asp Ser Pr - #o Val Asn Ile Glu Ala      385                 3 - #90                 3 - #95                 4 -      #00                                                                              - - Glu Pro Pro Phe Gly Gln Ser Tyr Ile Ile Il - #e Gly Val Glu Pro        Gly                                                                                             405  - #               410  - #               415             - - Gln Leu Lys Leu Asp Trp Phe Lys Lys Gly Se - #r Ser Ile Gly Gln Met                  420      - #           425      - #           430                  - - Phe Glu Thr Thr Met Arg Gly Ala Lys Arg Me - #t Ala Ile Leu Gly Asp              435          - #       440          - #       445                      - - Thr Ala Trp Asp Phe Gly Ser Lys Gly Gly Va - #l Phe Thr Ser Ile Gly          450              - #   455              - #   460                          - - Lys Ala Lys His Gln Val Phe Gly Ala Ile Ty - #r Gly Ala Ala Phe Ser      465                 4 - #70                 4 - #75                 4 -      #80                                                                              - - Gly Val Ser Trp Thr Met Lys Ile Leu Ile Gl - #y Val Ile Ile Thr        Trp                                                                                             485  - #               490  - #               495             - - Ile Gly Met Asn Ser Arg Ser Thr Ser Leu Se - #r Val Ser Leu Val Leu                  500      - #           505      - #           510                  - - Val Gly Ile Val Thr Leu Tyr Leu Gly Val Me - #t Val Gln Ala                      515          - #       520          - #       525                      - -  - - (2) INFORMATION FOR SEQ ID NO: 3:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH:  31 base - # pairs                                                (B) TYPE:  Nucleic A - #cid                                                   (C) STRANDEDNESS: Double                                                      (D) TOPOLOGY: Linear                                                 - -     (ii) SEQUENCE DESCRIPTION: SEQ ID NO: - #3:                           - - ACTGAGATCT  ATGATGGCCG  CAATCCTGGC A      - #                  - #              31                                                                      - -  - - (2) INFORMATION FOR SEQ ID NO: 4:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 31 base - #pairs                                                  (B) TYPE:  Nucleic A - #cid                                                   (C) STRANDEDNESS: Double                                                      (D) TOPOLOGY: Linear                                                 - -     (ii) SEQUENCE DESCRIPTION: SEQ ID NO: - #4:                           - - CTGACTGCAG  TTACGGCCTG  CACCATAACT  C     - #                  - #              31                                                                    __________________________________________________________________________

What is claimed is:
 1. An isolated and purified dengue virus DNAfragment consisting essentially of a DNA fragment which encodes acomplete dengue virus envelope protein and a carboxy terminus segment ofpremembrane protein which comprises a translocation signal for saidEnvelope protein.
 2. The isolated and purified DNA fragment according toclaim 1, wherein said dengue virus is dengue
 2. 3. The DNA fragment ofclaim 2 which encodes 495 amino acids of said envelope protein and 31amino acids of said carboxy terminus segment of premembrane protein,said fragment comprising the nucleotide sequence specified in SEQ ID NO:1 or an allelic variant which retains the neutralizing antibodyproduction characteristic of a protein encoded by SEQ ID No.
 1. 4. TheDNA fragment according to claim 3, wherein said DNA fragment encodes theamino acid sequence specified in SEQ ID NO:
 2. 5. The isolated andpurified DNA fragment according to claim 1, wherein said dengue isdengue
 1. 6. The isolated and purified DNA fragment according to claim1, wherein said dengue is selected from the group consisting of dengue 3and dengue
 4. 7. A recombinant DNA construct comprising:(i) a vector,and (ii) an isolated and purified dengue virus DNA fragment according toclaim
 1. 8. A recombinant DNA construct according to claim 7, whereinsaid dengue virus is dengue
 2. 9. The recombinant DNA constructaccording to claim 7, wherein said vector is a eukaryotic expressionvector.
 10. The recombinant DNA construct according to claim 8, whereinsaid vector is a eukaryotic expression vector.
 11. A recombinant DNAconstruct comprising:(i) a vector, and (ii) a dengue 2 DNA fragmentaccording to claim
 3. 12. The recombinant DNA construct according toclaim 11, wherein said vector is a eukaryotic expression vector.
 13. Therecombinant DNA construct according to claim 11, wherein said DNAfragment encodes the amino acids sequence specified in SEQ ID NO:
 2. 14.The recombinant DNA construct according to claim 11 wherein said vectoris pBlueBacIII.
 15. A host cell transformed with a recombinant DNAconstruct comprising:(i) a vector, and (ii) an isolated and purifieddengue virus DNA fragment according to claim
 1. 16. A host cellaccording to claim 15, wherein said cell is prokaryotic.
 17. The hostcell according to claim 15, wherein said cell is a eukaryotic cell. 18.A method for producing a dengue virus recombinant protein particle, saidmethod comprising the steps of:(i) culturing a host cell transformedwith an expression vector according to claim 9 under conditions suchthat said DNA fragment is expressed and said recombinant protein isproduced as a particle, said particle comprising more than one unit ofsaid recombinant protein; and (ii) isolating said recombinant proteinparticle.
 19. The method according to claim 18, wherein said denguevirus is dengue
 2. 20. The method of claim 18 wherein isolating saidrecombinant protein particle comprises:(i) pelleting said cells bycentrifugation, (ii) separating the cell pellet and the supernatant,(iii) lysing said cell pellet to release said recombinant proteinparticle; (iv) isolating said recombinant protein particle of step(iii); (v) fractionating said recombinant protein particle of step (iv)on a gradient; and (vi) isolating said recombinant protein particle, ina purified form.